Organisms must synthesize nucleotides in order for their cells to divide and replicate. Nucleotide synthesis in mammals may be achieved through one of two pathways: the de novo synthesis pathway; or the salvage pathway. Different cell types use these pathways to differing extents.
Inosine-5′-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) is an enzyme involved in the biosynthesis of guanine nucleotides. IMPDH catalyzes the NAD-dependent oxidation of inosine-5′-monophosphate (IMP) to xanthosine-5′-monophosphate (XMP) [Jackson R. C. et. al., Nature, 256, pp. 331-333, (1975)]. Regardless of species, the reaction involves the random addition of substrates. A conserved active site Cys residue attacks the C2 position of IMP and hydride is transferred to NAD+, producing NADH and the E-XMP* intermediate. NADH is released and a mobile flap folds into the vacant NADH site, E-XMP* hydrolyzes and XMP is released [W. Wang and L. Hedstrom, Biochemistry 36, pp. 8479-8483 (1997); J. Digits and L. Hedstrom, Biochemistry 38, pp. 2295-2306 (1999); Gan et al, Biochemistry 42, pp 847-863 (2003)]. The hydrolysis step is at least partially rate-limiting in most IMPDHs examined to date. The enzyme is unusual in that a large conformational change occurs in the middle of a catalytic cycle.
IMPDH is ubiquitous in eukaryotes, bacteria, archaebacteria, and protozoa [Y. Natsumeda & S. F. Carr, Ann. N.Y. Acad., 696, pp. 88-93 (1993)]. Two isoforms of human IMPDH, designated type I and type II, have been identified and sequenced [F. R Collart and E. Huberman, J. Biol. Chem., 263, pp. 15769-15772, (1988); Y. Natsumeda et al., J. Biol. Chem., 265, pp. 5292-5295, (1990)]. Type I has three isoforms derived from different mRNA splicing, with 514, 546 and 595 residues. Type II has 514 amino acids, and shares 84% sequence identity to the 514 isoform of Type I. Both IMPDH type I and type II form active tetramers in solution [Y. Yamada et al., Biochemistry, 27, pp. 2737-2745 (1988)].
Proliferation requires an expansion of the guanine nucleotide pool, so rapidly growing cells depend on IMPDH. Thus human IMPDHs are targets for anticancer chemotherapy [L. Che et al., Curr. Opin. Drug. Discov. Devel., 10, 403-12 92007); E. Olah et al., Adv. Enzyme. Regul., 46, 176-90 (2006)].
The activity of IMPDH is particularly important in B- and T-lymphocytes. These cells depend on the de novo, rather than salvage pathway to generate sufficient levels of nucleotides necessary to initiate a proliferative response to mitogen or antigen [A. C. Allison et. al., Lancet II, 1179, (1975) and A. C. Allison et al., Ciba Found. Symp., 48, 207, (1977)]. Thus, human IMPDHs are an attractive targets for selectively inhibiting the immune system without also inhibiting the proliferation of other cells.
Inhibitors of IMPDH are also known. U.S. Pat. No. 5,380,879 (incorporated by reference) and U.S. Pat. No. 5,444,072 (incorporated by reference) and PCT publications WO 94/01105 and WO 94/12184 describe mycophenolic acid (MPA) and some of its derivatives as potent, uncompetitive, reversible inhibitors of human IMPDH type I (Ki=33 nM) and type II (Ki=9 nM). MPA has been demonstrated to block the response of B- and T-cells to mitogen or antigen [A. C. Allison et. al., Ann. N.Y. Acad. Sci., 696, 63, (1993)].
Nucleoside analogs such as tiazofurin, ribavirin and mizoribine also inhibit IMPDH [L. Hedstrom, et. al. Biochemistry, 29, pp. 849-854 (1990); L. Hedstrom et al. Curr. Med. Chem. 1999, 6, 545-561]. These compounds require activation to either the adenine dinucleotide (tiazofurin) or monophosphate derivatives (ribavirin and mizoribine) that inhibit IMPDH. These activation pathways are often absent in the cell of interest. In addition, nucleoside analogs suffer from lack of selectivity and can be further metabolized to produce inhibitors of other enzymes. Therefore, nucleoside analogs are prone to toxic side effects.
Additionally, IMPDH has been shown to play a role in viral replication in some viral cell lines. [S. F. Carr, J. Biol. Chem., 268, pp. 27286-27290 (1993)]. Analogous to lymphocyte and tumor cell lines, the implication is that the de novo, rather than the salvage, pathway is critical in the process of viral replication.
IMPDH is also a promising target for antimicrobial chemotherapy. Microbial infections are now the second leading cause of death worldwide. Many commonly used antibiotics have been rendered ineffective by the upsurge of drug resistance, so there is an urgent need of new antimicrobial therapy. IMPDH2 is an essential gene in Mycobacterium tuberculosis, and deletion of IMPDH attenuates the virulence of many other bacteria. IMPDH inhibitors block the growth of Helicobacter pylori, Staphylococcus aureus, Candida albicans, Pneumocystis carinii, Leishmania donovani, Trypanosoma brucei gambienese, Eimeria tenella, Plasmodium falciparum and Cryptosporidium parvum in culture [L. Hedstrom et al., Curr. Med. Chem., 18, pp. 1909-1918 (2011)]. The prokaryotic IMPDHs share 30-40% sequence identity with the human enzyme, and have significantly different kinetic and functional properties. These observations indicate that specific inhibition of prokaryotic IMPDH can be achieved, and that such inhibitors are likely to have antibiotic activity. Curiously, Cryptospordium and several other eukaryotic organisms have prokaryote-like IMPDHs that appear to have been obtained via horizontal gene transfer. Eukaryotic organisms that contain a prokaryotic-like IMPDHs are also likely to be sensitive to prokaryotic IMPDH-specific inhibitors.
Cryptosporidiosis is a severe gastrointestinal disease caused by protozoan parasites of the genus Cryptosporidium. The most common causes of human disease are C. parvum and C. hominis, though disease can also result from C. felis, C. meleagridis, C. canis, and C. muris infection. Small children, pregnant women, the elderly, and immunocompromised people (e.g., AIDS patients) are at risk of severe, chronic and often fatal infection [Carey, C. M., Lee, H., and Trevors, J. T., Water Res., 38, 818-62 (2004); and Fayer, R., Veterinary Parasitology, 126, 37-56 (2004)]. Cryptosporidium infection is a major cause of diarrhea and malnutrition in the developing world. The Cryptosporidium parasites produce spore-like oocysts that are highly resistant to water chlorination. Several large outbreaks in the U.S. have been linked to drinking and recreational water. Infection rates are extremely high, with disease manifest in 30% of exposed individuals and a 50-70% mortality rate among immuno-compromised individuals. Furthermore, there is a growing and credible concern that these organisms could be deliberately introduced into the water supply in an act of bioterrorism. Effective drugs are urgently needed for the management of cryptosporidiosis in AIDS patients and/or epidemic outbreaks. Cryptosporidum parasites also cause significant disease in domestic livestock, especially calves, lambs, kids, foals, piglets and poultry.
All parasitic protozoa lack purine biosynthetic enzymes and must salvage purines from their hosts, making this pathway an extremely attractive target for developing antiprotozoal drugs. IMPDH is a key enzyme in the purine salvage pathway of C. parvum and general IMPDH inhibitors block parasite proliferation In vitro [N. N. Umejiego et al., J Biol Chem, 279 pp. 40320-40327 (2004); and B. Striepen et al, Proc Natl Acad Sci USA, 101 pp. 3154-9 (2004)]. The IMPDH protein of C. hominis is identical to that of C. parvum, as is the purine salvage pathway. As discussed above, IMPDH is a validated drug target in immunosuppressive, cancer and viral therapy, so the human enzymes are extremely well studied. Cryptospordium appears to have obtained its IMPDH gene from a proteobacterium. Thus C. parvum IMPDH has very different structure and properties than the human enzymes. IMPDHs from many pathogenic bacteria have similar structures to C. parvum IMPDH [Gollapalli et al., Chem. Biol., 17, 1084-1091 (2010)]. There is a need for selective IMPDH inhibitors that can slow or block parasite and bacterial proliferation. The present invention fulfills this need and has other related advantages.